Regulation of tissue healing by the extracellular matrix
Translated title
Regulering af vævsheling af den ekstracellulære matrix
Authors
Østergaard, Camilla Lyngsøe ; Gravesen, Emma Bust
Term
4. term
Publication year
2022
Submitted on
2022-06-01
Abstract
This thesis examines how the extracellular matrix (ECM) helps regulate tissue repair by comparing ECM produced by human adipose-derived stem cells (ASCs) and human foreskin fibroblasts, and by assessing how these ECM scaffolds influence macrophage behavior and immunophenotype. ASCs and fibroblasts were cultured under ECM-promoting conditions; the ECM was decellularized and characterized using qPCR of ECM-related genes (COL1, COL3, fibronectin, MMP1, MMP2, TIMP1, HAS2), immunofluorescence for collagen I/III, fibronectin, laminin and chondroitin sulfate, and Sirius Red/Fast Green to quantify collagen. CD14+ monocytes were differentiated into macrophage states (iM1, mM1, iM2, M2c), seeded onto ECM, and analyzed by morphology, picrosirius red staining, and flow-cytometric surface markers. Compared with fibroblast ECM, ASC ECM contained more total collagen, non-collagenous proteins and sulfated glycosaminoglycans, and more laminin, whereas fibroblast ECM showed higher levels of fibronectin, collagen I/III and chondroitin sulfate. Relative expression of COL1, COL3, fibronectin, MMP2, TIMP1 and HAS2 was higher in ASCs, while MMP1 was lower at day one. Macrophages displayed clear morphological differences on ECM, and surface marker profiles suggested that ASC-derived ECM may steer macrophages away from a pro-inflammatory phenotype. Overall, the findings indicate that ASCs deposit a richer ECM than fibroblasts and that ASC ECM may exert an immunomodulatory effect beneficial for wound healing.
Denne afhandling undersøger, hvordan den ekstracellulære matrix (ECM) bidrager til regulering af vævsheling ved at sammenligne ECM produceret af humane adipøse stamceller (ASC'er) og humane forhudsfibroblaster samt ved at vurdere, hvordan disse ECM-scaffolds påvirker makrofagers adfærd og immunfænotype. ASC'er og fibroblaster blev dyrket under betingelser, der fremmer ECM-deponering; ECM blev decellulariseret og karakteriseret med qPCR af ECM-relaterede gener (COL1, COL3, fibronectin, MMP1, MMP2, TIMP1, HAS2), immunfluorescens for kollagen I/III, fibronectin, laminin og chondroitinsulfat samt Sirius Red/Fast Green til kvantificering af kollagen. CD14+ monocytter blev differentieret til makrofagtilstande (iM1, mM1, iM2, M2c), seedet på ECM og analyseret ved morfologi, picrosiriusrød farvning og flowcytometrisk overflademarkørekspression. Sammenlignet med fibroblast-ECM indeholdt ASC-ECM mere samlet kollagen, ikke-kollagene proteiner og sulfaterede glycosaminoglykaner samt mere laminin, mens fibroblast-ECM havde højere niveauer af fibronectin, kollagen I/III og chondroitinsulfat. Relativ genekspression af COL1, COL3, fibronectin, MMP2, TIMP1 og HAS2 var højere i ASC'er, mens MMP1 var lavere dag ét. Makrofager udviste tydelige morfologiske forskelle på ECM, og overflademarkørprofiler indikerede, at ASC-afledt ECM kan dreje makrofager væk fra en pro-inflammatorisk fænotype. Samlet peger resultaterne på, at ASC'er deponerer en rigere ECM end fibroblaster, og at ASC-ECM kan udøve en immunmodulerende indflydelse, der potentielt er gunstig for sårheling.
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